Method for directional cloning

ABSTRACT

Described herein are techniques for directional cloning an insert DNA segments into a target vector. The techniques mix the target vector, the insert DNA segment, a restriction enzyme, and a DNA ligase to generate a recombinant DNA molecule.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International applicationnumber PCT/CN2014/088800, titled “Method for Directional Cloning,” filedon Oct. 17, 2014, which is hereby incorporated by reference.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification.

TECHNICAL FIELD

This disclosure relates to recombinant technologies. More specifically,the disclosure relates to methods for directional cloning.

BACKGROUND

Molecular cloning is a process used to create recombinant DNA molecules.Components for the formation of recombinant DNA include a cloningvector, a small DNA segment, multiple enzymes, and living cells. Thecloning vector is a DNA molecule that replicates within the livingcells. The small DNA segment contains a gene to be cloned. During thecloning process, the gene may be combined with the vector using variousmethods to form the recombinant DNA molecules. The recombinant DNAmolecules are then transformed into the living cells, which are thenscreened and duplicated. However, conventional techniques for molecularcloning involve multiple processes under various conditions. Therefore,there is a need for a simple, efficient, inexpensive method of molecularcloning of a DNA segment into a target molecule.

SUMMARY

Described herein are methods for directional cloning of an insert DNAsegment into a vector. The various embodiments including providing atarget vector and an insert Deoxyribonucleic acid (DNA) segment. Thetarget vector and the insert DNA segment may be mixed with an amount ofa restriction enzyme, and an amount of a DNA ligase in a container tocleave the target vector at a first temperature and to ligate at leastone portion of the insert DNA segment to the target vector at a secondtemperature. In some embodiments, the first temperature is the same orsubstantially the same as the second temperature.

In some embodiments, the insert DNA segment to the target vector may beligated to generate a recombinant DNA molecule including at least oneportion of the target vector and the at least one portion of the insertDNA segment. In these instances, the recombinant DNA is an expressionvector. In some embodiments, the insert DNA segment may include the atleast one portion of the insert DNA segment and DNA sequences includingrecognition sites of the restriction enzyme. In some embodiments, thetarget vector may include recognition sites of the restriction enzyme(e.g., a type II restriction enzyme).

In some embodiments, molecules of the target vector in the containersubstantially are closed circular plasmids when the target vector, theinsert DNA segment, the amount of the restriction enzyme, the amount ofthe DNA ligase are mixed. In some embodiments, the target vector, theinsert DNA segment, the amount of the restriction enzyme, and the amountof a DNA ligase may be mixed in a single container for a predeterminedtime period. For example, the predetermined time period may include atime period of from about 1 minutes to 20 minutes.

In some embodiments, the restriction enzyme may include a type IIrestriction enzyme. In these instances, the type II restriction enzymemay include at least one of BsaI, BbsI, BsmBI, Alw26I or LguI. In someembodiments, the DNA ligase may include at least one of T4 DNA ligase orE. coli DNA ligase.

In some embodiments, the Insert DNA segment may include a polymerasechain reaction (PCR) product. In some embodiments, the first temperatureand/or the second temperature is a predetermined temperature. Forexample, the predetermined temperature may range from about 16° C. toabout 37° C.

Some embodiments relate to the directional cloning of multiple DNAfragments. These embodiments enable Simple, fast, efficient,directional, and seamless directional cloning for DNA transduction. Forexample, some embodiments may enable the PCR product to be cloned intoexpression vectors, while eliminating the need for an intermediateprocess such as cloning into T vector (See FIG. 3 and FIG. 4).

Some embodiments relate to a method for directional cloning. The methodmay include providing a plurality of DNA segments for the directionalcloning, incubating a lentiviral vector, the plurality of DNA segments,restriction enzymes, and DNA ligases in a single container at a roomtemperature for a period less than 30 minutes to obtain the lentiviralvector including a recombinant DNA molecule may include the plurality ofDNA segments in the desired direction, and mixing the lentiviral vectorwith a competent host cell.

In some embodiments, a positive rate of the directional cloning isgreater than 80%.

In some embodiments, the number of the plurality of DNA segments is morethan 2.

In some embodiments, wherein comprises amplifying DNA segments bypolymerase chain reaction (PCR) to obtain the plurality of DNA segments,respectively, and the plurality of DNA segment are a plurality ofmodified DNA segment.

In some embodiments, wherein the restriction enzyme comprises a type IIrestriction enzyme.

In some embodiments, the restriction enzymes comprise at least one ofBsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, orLguI.

In some embodiments, the DNA ligases comprise at least one of T4 DNAligase or E. coli DNA ligase.

In some embodiments, an individual DNA segment of the plurality of DNAsegments may include adaptors, and the recombinant DNA molecule does notcomprise adaptors.

In some embodiments, the period is less than 15 minutes.

In some embodiments, the period is about 10 minutes.

Some embodiments relate to a reaction mixture for use in directionalcloning; the reaction mixture may include a plurality of DNA segments, alentiviral vector, a plurality of amplified DNA segments for thedirectional cloning, restriction enzymes, and DNA ligases. In theseinstances, the number of the plurality of DNA segments is more than 2,and the directional cloning is performed at a room temperature for aperiod less than 30 min.

In some embodiments, a positive rate of the directional cloning isgreater than 80%.

In some embodiments, the number of the plurality of DNA segments is morethan 2.

In some embodiments, comprises amplifying DNA segments by polymerasechain reaction (PCR) to obtain the plurality of DNA segments,respectively, and the plurality of DNA segment are a plurality ofmodified DNA segment.

In some embodiments, the restriction enzyme comprises a type IIrestriction enzyme.

In some embodiments, the restriction enzymes comprise at least one ofBsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, orLguI.

In some embodiments, the DNA ligases comprise at least one of T4 DNAligase or E. coli DNA ligase.

In some embodiments, an individual DNA segment of the plurality of DNAsegments may include adaptors.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify all key featuresor essential features of the claimed subject matter, nor is it intendedto be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description is described with reference to the accompanyingfigures.

FIG. 1 is a diagram showing an exemplary scheme for directional cloningof an insert DNA segment into a vector.

FIG. 2 is a diagram showing an exemplary plasmid.

FIG. 3 includes diagrams illustrating the preparation of PCR products.

FIG. 4 is a diagram illustrating directional cloning of PCR products.

FIG. 5 illustrates the construction of an expression vector (beforereaction): Lv-ef1 α-CCDB-IRES-EGFP and Lv-ef1 α-CCDB. Region 502(replacement region) represents a region of the fragment will bereplaced after the reaction. Replacement region 502 includes Lv-ef1α-CCDB-IRES-EGFP 7733-9176 or Lv-ef1 α-CCDB 6426-7869.

FIG. 6 shows the construction of Lv-ef1 α-luciferase-IRES-EGFP SEQ IDNo: 12 and Lv-ef1 α-luciferase SEQ ID NO: 13.

FIG. 7 shows the construction of three fragments.

FIG. 8 illustrates single fragment cloning.

FIG. 9 illustrates directional cloning of three fragments.

DETAILED DESCRIPTION Definition

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotidehaving a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor encoding an amino acid sequence identical to an amino acid sequencein a peptide or protein; or (b) a peptide or polypeptide having an aminoacid sequence that is substantially identical to a sequence of aminoacids in a reference peptide or protein.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example by conjugation or complexingwith other chemical moieties (e.g., pegylation) or by post-translationalmodification techniques as would be understood in the art. The term“derivative” is also meant a chemical compounds (e.g., sugars) that hasbeen derived from the basic structure by modification, for example, byconjugation or complexing with other chemical moieties (e.g.,glycosylation). The term “derivative” also includes within its scopealterations that have been made to a parent sequence/structure includingadditions or deletions that provide for functionally equivalentmolecules.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395) which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a recombinanthost cell.

By “isolated” is meant a material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide”, as used herein, refers to apolynucleotide, which has been purified from the sequences which flankit in a naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, refer to in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell.

The terms “modulating” and “altering” include “increasing” and“enhancing” as well as “decreasing” or “reducing,” typically in astatistically significant or a physiologically significant amount ordegree relative to control. In specific embodiments, immunologicalrejection associated with transplantation of the blood substitutes isdecreased relative to an unmodified or differently modified stem cell byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 150%, at least 200%, at least 300%,at least 400%, at least 500%, or at least 1000%.

An “increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 1.3,1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein.

A “decreased” or “reduced” or “lesser” amount is typically a“statistically significant” amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein. For example, “In specific embodiments, immunologicalrejection associated with transplantation of the blood substitutes isdecreased relative to an unmodified or differently modified stem cell byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 150%, at least 200%, at least 300%,at least 400%, at least 500%, or at least 1000%.

By “obtained from” is meant that a sample such as, for example, apolynucleotide or polypeptide is isolated from, or derived from, aparticular source, such as the desired organism or a specific tissuewithin the desired organism. “Obtained from” can also refer to thesituation in which a polynucleotide or polypeptide sequence is isolatedfrom, or derived from, a particular organism or tissue within anorganism. For example, a polynucleotide sequence encoding a referencepolypeptide described herein may be isolated from a variety ofprokaryotic or eukaryotic organisms, or from particular tissues or cellswithin a certain eukaryotic organism.

The recitation “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refersto a polymeric form of nucleotides of at least 10 bases in length,either ribonucleotides or deoxynucleotides or a modified form of eithertype of nucleotide. For example, polynucleotides may include singleand/or double-stranded forms of DNA molecules and RNA molecules.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

As used herein, the term “a polynucleotide having a nucleotide sequenceencoding a gene” means a nucleic acid sequence containing the codingregion of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in either acDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the vectors ofthe present disclosure may contain endogenous enhancers/promoters,splice junctions, intervening sequences, polyadenylation signals, etc.or a combination of both endogenous and exogenous control elements.

A nucleic acid sequence refers to a series of nucleotides, which isrepresented by a succession of letters that indicate the order of thenucleotides within a DNA (using G, A, C, and T) or an RNA (using G, A,C, and U) molecule. By convention, a nucleic acid sequence is usuallypresented from the 5′ end to the 3′ end. For example, a DNA sequencerefers to a series of nucleotides connected one to the other byphosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule including multiple segments of DNA joinedtogether by means of molecular biological techniques. The term“recombinant protein” or “recombinant polypeptide” as used herein refersto a protein molecule which is expressed from a recombinant DNAmolecule.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize to areference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletions,and substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide or has increased activity in relation tothe reference polynucleotide (i.e., optimized). Polynucleotide variantsinclude, for example, polynucleotides having at least 50% (and at least51% to at least 99% and all integer percentages in between, e.g., 90%,95%, or 98%) sequence identity with a reference polynucleotide sequencedescribed herein. The terms “polynucleotide variant” and “variant” alsoinclude naturally-occurring allelic variants and orthologs that encodethese enzymes.

With regard to polynucleotides, the term “exogenous” refers to apolynucleotide sequence that does not naturally occur in a wild-typecell or organism but is typically introduced into the cell by molecularbiological techniques. Examples of exogenous polynucleotides includevectors, plasmids, and/or man-made nucleic acid constructs encoding thedesired protein. With regard to polynucleotides, the term “endogenous”or “native” refers to naturally-occurring polynucleotide sequences thatmay be found in a given wild-type cell or organism. Also, a particularpolynucleotide sequence that is isolated from a first organism andtransferred to the second organism by molecular biological techniques istypically considered an “exogenous” polynucleotide with respect to thesecond organism. In specific embodiments, polynucleotide sequences canbe “introduced” by molecular biological techniques into a microorganismthat already contains such a polynucleotide sequence, for instance, tocreate one or more additional copies of an otherwise naturally-occurringpolynucleotide sequence, and thereby facilitate overexpression of theencoded polypeptide.

“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes,” whichtypically catalyze (i.e., increase the rate of) various chemicalreactions.

The term “reference sequence” generally refers to a nucleic acid codingsequence, or amino acid sequence, to which another sequence is beingcompared. All polypeptide and polynucleotide sequences described hereinare included as references sequences.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence. For example, an “EcoR”and “BamH” cut a DNA molecule at recognition sites. As used herein,“recognition site” refers to a sequence of specific bases that isrecognized by a restriction enzyme if the sequence is present indouble-stranded DNA; or, if the sequence is present in single-strandedRNA, the sequence of specific bases that would be recognized by arestriction enzyme if the RNA was reverse transcribed into cDNA and thecDNA employed as a template with a DNA polymerase to generate adouble-stranded DNA; or, if the sequence is present in single-strandedDNA, the sequence of specific bases that would be recognized by arestriction enzyme if the single-stranded DNA was employed as a templatewith a DNA polymerase to generate a double-stranded DNA; or, if thesequence is present in double-stranded RNA, the sequence of specificbases that would be recognized by a restriction enzyme if either strandof RNA was reverse transcribed into cDNA and the cDNA employed as atemplate with a DNA polymerase to generate a double-stranded DNA. Theterm “unique restriction enzyme site” indicates that the recognitionsequence for a given restriction enzyme appears once within a nucleicacid molecule.

A DNA Ligase refers to an enzyme that creates a phosphodiester bondbetween the 3′ end of one DNA segment and the 5′ end of another, whilethey are base-paired to a template strand. For example, DNA ligaserepairs the ends of single-stranded DNA in a duplex DNA chain and/orrepair double-strand breaks.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% sequence identity to any of the reference sequences describedherein (see, e.g., Sequence Listing), typically where the polypeptidevariant maintains at least one biological activity of the referencepolypeptide.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 90%, 95%, 96%, 97%, 98%, 99% or greater of some givenquantity.

“Transformation” refers to the permanent, heritable alteration in a cellresulting from the uptake and incorporation of foreign DNA into thehost-cell genome; also, the transfer of an exogenous gene from oneorganism into the genome of another organism.

As used herein, the terms “selectable marker” or “selectable markergene” refers to the use of a gene which encodes an enzymatic activitythat confers the ability to grow in medium lacking what would otherwisebe an essential nutrient (e.g., the TRP1 gene in yeast cells); inaddition, a selectable marker may confer resistance to an antibiotic ordrug upon the cell in which the selectable marker is expressed. Aselectable marker may be used to confer a particular phenotype upon ahost cell. When a host cell must express a selectable marker to grow inselective medium, the marker is said to be a positive selectable marker(e.g., antibiotic resistance genes which confer the ability to grow inthe presence of the appropriate antibiotic). Selectable markers can alsobe used to select against host cells containing a particular gene (e.g.,the sacB gene which, if expressed, kills the bacterial host cells grownin medium containing 5% sucrose); selectable markers used in this mannerare referred to as negative selectable markers or counter-selectablemarkers.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast orvirus, into which a polynucleotide can be inserted or cloned. A vectormay contain one or more unique restriction sites and can be capable ofautonomous replication in a defined host cell including a target cell ortissue or a progenitor cell or tissue thereof, or be integrable with thegenome of the defined host such that the cloned sequence isreproducible. Accordingly, the vector can be an autonomously replicatingvector, i.e., a vector that exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g., alinear or closed circular plasmid, an extra-chromosomal element, amini-chromosome, or an artificial chromosome. The vector can contain anymeans for assuring self-replication. Alternatively, the vector can beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated. Such a vector may comprise specific sequences thatallow recombination into a particular, desired site of the hostchromosome. A vector system can comprise a single vector or plasmid, twoor more vectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the host cell into which the vector is to be introduced. In thepresent case, the vector is preferably one which is operably functionalin a stem cell. The vector can include a reporter gene, such as a greenfluorescent protein (GFP), which can be either fused in frame to one ormore of the encoded polypeptides or expressed separately. The vector canalso include a selection marker such as an antibiotic resistance genethat can be used for selection of suitable transformants.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “PCR product” refers to deoxynucleosidetriphosphate(s) copies derived from a DNA template using a polymerasechain reaction (PCR)-based amplification.

Embodiments

Various embodiments relate to methods for directional cloning, whichallows an insert DNA to be ligated to a vector in a specific orientationand/or prevent the vector from self-ligation. As defined herein,directional cloning refers to a procedure designed to ensure that aninsert DNA is inserted into a target vector molecule in a definiteand/or known orientation. The directional cloning may be needed, forexample, when the insert DNA is subsequently to be transcribed from apromoter sequence within the target vector.

Under conventional techniques related to the directional cloning,multiple thermal cycles are performed using a PCR machine to generatethe desired recombinant target vector. In some instances, sub-cloning isalso performed to identify the desired recombined target vector.Embodiments herein related to a surprising or unexpected discovery thatcleaving of a target vector and ligating one or more DNA insert DNAsegments to the target vector may be performed at substantially the sametemperature to form a recombined target vector.

As illustrated in FIG. 1, a method may include providing a target vector102, and an insert DNA segment 104 at 106. The target vector 102 and theinsert DNA segment 104 may be mixed with an amount of a restrictionenzyme and an amount of a DNA ligase in a container 108 at 110. In someembodiments, the target vector 102 may be digested and cleaved by therestriction enzyme and at least one portion of the insert DNA segment104 may be ligated to the cleaved target vector 102 to form arecombinant DNA molecule 112 at 114. In these instances, the at leastone portion of the insert DNA segment 104 is represented by an insertedDNA segment 116.

In some embodiments, the recombinant DNA molecule is an expressionvector, which may include at least one portion of the target vector andat least one portion of the insert DNA segment.

In some embodiments, the insert DNA segment 104 may include the insertedDNA segment 116 and one or more DNA sequences including recognitionsites of the restriction enzyme. For example, the 5′ end of the insertDNA segment 104 may include a recognition site of the restriction enzymesuch that the recognition site may be removed by the restriction enzymeand the inserted DNA segment 116 may be ligated to the cleaved targetvector 102 to form the recombinant DNA molecule 112. In someembodiments, the insert DNA segment 104 may be generated by PCR-basedamplification reactions. In some embodiments, the target vector 102 mayinclude recognition sites of the restriction enzyme (e.g., a type IIrestriction enzyme).

In some embodiments, the target vector 102, the insert DNA segment 104,the restriction enzyme, and the DNA ligase may be mixed in the container108, for example, a microcentrifuge tube. In these instances, the targetvector 102 and/or insert DNA segment 104 may be digested and cleaved bythe restriction enzyme at a first temperature. The first temperature isthe same or substantially the same as a second temperature at which theinserted DNA segment 116 is ligated to the cleaved target vector 102.

In some embodiments, the target vector 102, the insert DNA segment 104,the restriction enzyme, and the DNA ligase may be mixed in the container108. In these instances, the molecules of the target vector 102 in thecontainer 108 substantially are closed circular plasmids before themolecules of the target vector 102 are digested and cleaved by therestriction enzyme in the container 108. In some embodiments, unlikeconventional techniques, the embodiments of the present disclosure mayperform the digestion using the restriction enzyme and the ligationusing the DNA ligase under the same one or more condition parameters.The condition parameters may include a reaction temperature, a reactiontime period, and/or an amount of enzyme usage. For example, the targetvector 102, the insert DNA segment 104, the restriction enzyme, and theDNA ligase may be mixed in the container 108 at a room temperature andincubated for 10 minutes, such that the digestion and ligation may beperformed under the same condition parameters.

In some embodiments, the target vector 102, the insert DNA segment 104,the amount of the restriction enzyme, and the amount of a DNA ligase maybe mixed in the container 108 for a time period of from about 1 minutesto 20 minutes at a temperature of from about 16° C. to about 37° C.

In some embodiments, the restriction enzyme may include a type IIrestriction enzyme. For example, the type II restriction enzyme mayinclude BsaI, BbsI, BsmBI, Alw26I, or LguI. In some embodiments, the DNAligase may include T4 DNA ligase or E. coli DNA ligase.

In some embodiments, the target vector 102, multiple copies of theinsert DNA segment 104, an amount of a restriction enzyme, and an amountof a DNA ligase may be mixed in the container 108 to cleave the targetvector 102 and/or the multiple copies of the insert DNA segment 104 atthe first temperature, and to ligate multiple copies of at least oneportion of the insert DNA segment to the target vector at a secondtemperature. In some instances, the first temperature is the same orsubstantially the same as a second temperature.

In some embodiments, the target vector 102, the insert DNA segment 104,an additional insert DNA segment, an amount of a restriction enzyme, andan amount of a DNA ligase may be mixed to cleave the target vector 102,the insert DNA segment 104, and the additional insert DNA segment at afirst temperature, and to ligate at least one portion of the insert DNAsegment and at least one portion of the additional insert DNA segment tothe target vector at a second temperature. In some instances, a sequenceof the insert DNA segment 102 may be different from a sequence of theadditional insert DNA segment. In some instances, the first temperatureis the same or substantially the same as a second temperature.

Some embodiments relate to the directional cloning of multiple DNAfragments. These embodiments enable Simple, fast, efficient,directional, and seamless directional cloning for DNA transduction. Forexample, some embodiments may enable the PCR product to be cloned intoexpression vectors, while eliminating the need for an intermediateprocess such as cloning into T vector (See FIG. 3 and FIG. 4).

Some embodiments relate to a method for directional cloning. The methodmay include providing a plurality of DNA segments for the directionalcloning, incubating a lentiviral vector, the plurality of DNA segments,restriction enzymes, and DNA ligases in a single container at a roomtemperature for a period less than 30 minutes to obtain the lentiviralvector including a recombinant DNA molecule may include the plurality ofDNA segments in the desired direction, and mixing the lentiviral vectorwith a competent host cell.

In some embodiments, a positive rate of the directional cloning isgreater than 80%.

In some embodiments, the number of the plurality of DNA segments is morethan 2.

In some embodiments, wherein comprises amplifying DNA segments bypolymerase chain reaction (PCR) to obtain the plurality of DNA segments,respectively, and the plurality of DNA segment are a plurality ofmodified DNA segment.

In some embodiments, wherein the restriction enzyme comprises a type IIrestriction enzyme.

In some embodiments, the restriction enzymes comprise at least one ofBsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, orLguI.

In some embodiments, the DNA ligases comprise at least one of T4 DNAligase or E. coli DNA ligase.

In some embodiments, an individual DNA segment of the plurality of DNAsegments may include adaptors, and the recombinant DNA molecule does notcomprise adaptors.

In some embodiments, the period is less than 15 minutes.

In some embodiments, the period is about 10 minutes.

Some embodiments relate to a reaction mixture for use in directionalcloning; the reaction mixture may include a plurality of DNA segments, alentiviral vector, a plurality of amplified DNA segments for thedirectional cloning, restriction enzymes, and DNA ligases. In theseinstances, the number of the plurality of DNA segments is more than 2,and the directional cloning is performed at a room temperature for aperiod less than 30 min.

In some embodiments, a positive rate of the directional cloning isgreater than 80%.

In some embodiments, the number of the plurality of DNA segments is morethan 2.

In some embodiments, comprises amplifying DNA segments by polymerasechain reaction (PCR) to obtain the plurality of DNA segments,respectively, and the plurality of DNA segment are a plurality ofmodified DNA segment.

In some embodiments, the restriction enzyme comprises a type IIrestriction enzyme.

In some embodiments, the restriction enzymes comprise at least one ofBsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, orLguI.

In some embodiments, the DNA ligases comprise at least one of T4 DNAligase or E. coli DNA ligase.

In some embodiments, an individual DNA segment of the plurality of DNAsegments may include adaptors.

EXAMPLES Example 1 Preparation of Insert DNA Segments

An insert DNA segment carried the desired insert sequence (SEQ ID NO: 1)and restriction sites (SEQ ID NO: 2) that were recognized by a BsaIrestriction enzyme. The restriction sites were located on the 5′ and 3′ends of the insert DNA. The insert DNA was amplified by the polymerasechain reaction (PCR). Sequences of the forward and reverse primersdesigned for the PCR and other sequences are provided in Table 1.Polymerases used in the PCR included Taq or a high fidelity DNApolymerase. After the amplification, the PCR products were purifiedusing enzymes, such as DpnI and/or DMT.

TABLE 1 Name SEQ ID Desired Insert Sequence SEQ ID NO: 1 RestrictionSite SEQ ID NO: 2 Forward Primer SEQ ID NO: 3 Reverse Primer SEQ ID NO:4

Example 2 Generation of Expression Vectors

1-50 nanogram (ng) of the purified insert DNA segments were mixed with25 ng of pSDS201 plasmid, 10× buffer, an enzyme mixture including 20 Urestriction enzyme and 5 U T4 DNA ligase (e.g., 1 ul restriction enzymeand 1 ul T4 DNA ligase), and double-distilled water (ddH2O) in a singletube. More detailed information is presented in Table 2. The pSDS201plasmid is about 4600 bp and includes multiple restriction sites ofBsaI. As shown in FIG. 2, construct 200 illustrates construct of apSDS201 plasmid. The mixture was incubated at a temperature of from 22°C. to 37° C. for 10 minutes to obtained reaction products includingrecombined DNA molecules. An individual recombined DNA molecule of therecombined DNA molecules include the desired insert sequence and theplasmid.

TABLE 2 Components Volume Plasmids 25 ng Inserts 1-50 ng 10x Buffer 2 μlEnzyme Mix 2 μl ddH₂O Up to 20 μl Total 20 μl

Example 3 Transformation and Assay of the Expression Vectors

After the incubation, 10 μl of the reaction products were mixed with 100μl solution containing 10⁸ units of DH5α competent cells to obtainvarious transformants. These transformants were first screened based ona selectable marker (e.g., Kana) in the pSDS201 plasmids. Colonyscreening methods were further performed to select the transformantincluding expression vectors containing the recombined DNA molecules.The colony screening methods included colony PCR, restrictionendonuclease digestion, and/or DNA sequencing.

Example 4 Directional Cloning of Single and Multiple DNA Fragments

Two specific PCR primers were added by one or more specific adaptors,and the PCR produces and vectors were mixed with enzymes for about 10min at the room temperature such that directional cloning can be carriedout. The positive rate is greater than 80%.

Without purification using enzymes (e.g., (DNase)), insert PCR productswere used directly in DNA Cloning. For example, such directional cloningeliminates the need for recycling purified by gel digestion step andsimplifies the experimental procedure to save time. In some instances,the vector retains double digestion sites.

PCR products were prepared using the operations as follow. Based on theprinciples of primer design, validation purposes fragment does notcontain a sequence “GGTCTC.”

Further, target fragments were designed, and PCR primers includedspecific primer of target fragments and specific adaptors. Primers oftarget fragments were designed based on principles of primer designs.

In some instance, if the PCR product is an amplified template plasmidand the recombinant plasmid vector and has the same resistance, it isrecommended that the plasmid template PCR products were digested withDpn1. The PCR products included multiple inserts for directional cloningusing the configuration provided in Table 2.

TABLE 2 Reaction System Configuration Vector: 50 ng Insert: 1-50 ng 10XT4 DNA ligase buffer 2 ul T4 DNA ligase 0.5 ul Bsa1 0.5 ul sterile waterto make up to 20 ul

After the system configuration is complete, the reaction mixture wasmixed using a pipette to avoid air bubbles. The reaction mixture wasincubated at a room temperature (e.g., 10-37° C.) for about 10 minutes.After the incubation, the reaction mixture may be directly used fortransduction or stored at −20° C.

In some cases, the following protocol was also used for directionalcloning of multiple inserts. The reaction mixture was incubated at 37°C. for 5 minutes and then 22° C. for 5 minutes for 15 cycles, and at 37°C. 30 minutes.

The recombinant product was transformed and plated. 10 ul ligationproduct was taken and added to 100 ul competent cells, mixed, on ice for30 min, and then heat shock 42° C. 45 s, ice-water bath for 5 minute.500 ul SOC medium was added, 37° C. 30 min shaking bacteria, the mixturewas centrifuged 5 min at 4000 rpm (1.5 g). The supernatant wasdiscarded, the pellet was resuspended leaving 100 ul coating onampicillin-containing plates to 50 ug/ml, and the plates were invertedand incubated 12-16 h at 37° C. C. Identification of positive clones wasthen performed. 2-3 bacterial clones were randomly selected andinoculated into ampicillin broth 50 ug/ml of bacterial cultureovernight. Plasmids were extracted and sequenced.

FIG. 5 illustrates the construction of an expression vector (beforereaction): Lv-ef1 α-CCDB-IRES-EGFP and Lv-ef1 α-CCDB. Region 502(replacement region) represents a region of the fragment will bereplaced after the reaction. Replacement region 502 includes Lv-ef1α-CCDB-IRES-EGFP 7733-9176 or Lv-ef1 α-CCDB 6426-7869.

Taking luciferase as an example, following sequences may be used fordirectional cloning using Luciferase of which sequence is of SEQ ID No:5. Directional cloning of a single fragment was performed using thefollowing sequences in Table 3 and conditions described above. FIG. 6shows the construction of Lv-ef1 α-luciferase-IRES-EGFP SEQ ID No: 12and Lv-ef1 α-luciferase SEQ ID NO: 13.

TABLE 3 Sequences for directional cloning of a single fragment Primersequence of Insert luciferase SEQ ID No: 6: forward primer: 5′ SEQ IDNo: 7: a Reverse primer: 5′ Specific primer of target fragment of insertluciferase SEQ ID No: 8: forward primer: 5′ SEQ ID No: 9: Reverseprimer: 5′ Adaptor of insert luciferase SEQ ID No: 10: adaptor Forward:5′ SEQ ID No: 11: Reverse adaptor: 5′

Directional cloning of three fragments was performed using the followingsequences in Table 4 and conditions described above. Sequences of threesegments were connected and inserted into the vector (FIGS. 7 and 9).After processed described above, inserts 1, 2, and 3 are inserted intothe vector to obtain a directional cloning product. The positive rate isgreater than 80%.

TABLE 3 Sequences for directional cloning of three fragments Insert 1:1-419bp of SEQ ID NO 5 Insert 2: 420-1172bp of SEQ ID NO 5 Insert 3:1173-1653bp of SEQ ID NO 5 Primer sequence of insert 1 SEQ ID NO: 14Forward primer: 5′ SEQ ID NO: 15 Reverse primer: 5′ Specific primer oftarget fragment of insert 1 SEQ ID NO: 16 Forward primer: 5′ SEQ ID NO:17 Reverse primer: 5′ Adaptor of insert 1 SEQ ID NO: 18: adaptorForward: 5′ SEQ ID NO: 19: Reverse adaptor: 5 (the four bases are notlocated on insert 1 and are not listed) Primer sequence of insert 2 SEQID NO: 20: Forward primer: 5′ SEQ ID NO: 21: Reverse primer: 5′ Specificprimer of target fragment of insert 2 SEQ ID NO: 22: Forward primer: 5′SEQ ID NO: 23: Reverse primer: 5′ Adaptor of insert 2 SEQ ID NO: 24:adaptor Forward: 5′ SEQ ID NO: 25: Reverse adaptor: 5′(a single C oninsert 2 and is not listed) Primer sequence of insert 3 SEQ ID NO: 26:Forward primer: 5′ SEQ ID NO: 27: Reverse primer: 5′ Specific primer oftarget fragment of insert 3 SEQ ID NO: 28: Forward primer: 5′ SEQ ID NO:29: Reverse primer: 5′ Adaptor of insert luciferase SEQ ID NO: 30:adaptor Forward: 5′(tgt are located on the insert 3 and not listed) SEQID NO: 31: Reverse adaptor: 5′

What is claimed is:
 1. A method for directional cloning, the methodcomprising: providing a plurality of DNA segments for the directionalcloning; incubating a lentiviral vector, the plurality of DNA segments,restriction enzymes, and DNA ligases in a single container at a roomtemperature for a period less than 30 minutes to obtain the lentiviralvector comprising a recombinant DNA molecule comprising the plurality ofDNA segments in a desired direction; and mixing the lentiviral vectorwith a competent host cell.
 2. The method of claim 1, wherein a positiverate of the directional cloning is greater than 80%.
 3. The method ofclaim 2, wherein the number of the plurality of DNA segments is morethan
 2. 4. The method of claim 1, wherein comprises amplifying DNAsegments by polymerase chain reaction (PCR) to obtain the plurality ofDNA segments, respectively, and the plurality of DNA segment are aplurality of modified DNA segment.
 5. The method of claim 1, wherein therestriction enzyme comprises a type II restriction enzyme.
 6. The methodof claim 5, wherein the restriction enzymes comprise at least one ofBsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, orLguI.
 7. The method of claim 1, wherein the DNA ligases comprise atleast one of T4 DNA ligase or E. coli DNA ligase.
 8. The method of claim1, wherein an individual DNA segment of the plurality of DNA segmentscomprising adaptors, and the recombinant DNA molecule does not compriseadaptors.
 9. The method of claim 1, wherein the period is less than 15minutes.
 10. The method of claim 1, wherein the period is about 10minutes.
 11. A reaction mixture for use in directional cloning, thereaction mixture comprising a plurality of DNA segments, a lentiviralvector, a plurality of amplified DNA segments for the directionalcloning, restriction enzymes, and DNA ligases, wherein the number of theplurality of DNA segments is more than 2, and the directional cloning isperformed at a room temperature for a period less than 30 min.
 12. Thereaction mixture of claim 11, wherein a positive rate of the directionalcloning is greater than 80%.
 13. The reaction mixture of claim 12,wherein the number of the plurality of DNA segments is more than
 2. 14.The reaction mixture of claim 11, wherein comprises amplifying DNAsegments by polymerase chain reaction (PCR) to obtain the plurality ofDNA segments, respectively, and the plurality of DNA segment are aplurality of modified DNA segment.
 15. The reaction mixture of claim 11,wherein the restriction enzyme comprises a type II restriction enzyme.16. The reaction mixture of claim 15, wherein the restriction enzymescomprise at least one of BsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI,BspMI, BtgZI, SfaNI, or LguI.
 17. The reaction mixture of claim 11,wherein the DNA ligases comprise at least one of T4 DNA ligase or E.coli DNA ligase.
 18. The reaction mixture of claim 11, wherein anindividual DNA segment of the plurality of DNA segments comprisingadaptors.